This invention relates to cryopreservation and long-term storage of cultured epithelial tissue sheets useful as skin wound dressings in a manner which maintains cell viability and colony-forming efficiency.
It has been a priority in the medical community to develop a skin wound dressing which will encourage new cell growth while preventing fluid loss and infection following skin wounds from burns, ulceration, or surgical excision. Since traditional dressings fail to protect large-scale wounds adequately, several alternatives have been developed. Among these alternatives are split- and full-thickness grafts of cadaver skin, porcine skin, and human allografts and autografts. Most have proved unsatisfactory since all but autografts eventually are rejected by the body in the absence of immunosuppressive therapy. In addition, use of conventional autografting techniques is not practical for massive burn injury involving large body surface areas.
Green et al. have developed a method of culturing epithelial cell sheets several cells thick for repairing burns, ulcerations and other skin wounds. U.S. Pat. No. 4,016,036 discloses the method for serially culturing keratinocytes to produce stratified sheets of epithelium. U.S. Pat. No. 4,304,866 discloses the method of producing transplantable cell sheets by culturing keratinocytes and detaching the sheet from its anchorage substrate using an enzyme such as dispase. U.S. Pat. No. 4,456,687 discloses agents useful to promote growth of epithelial cells. The disclosures of these patents are incorporated herein by reference. In the culture system developed by Green et al., epithelial cells divide rapidly on the surface of tissue culture dishes or flasks, and ultimately form a confluent, modestly stratified sheet of tightly interconnected cells. These confluent cultures can be released as a cohesive cell sheets by treatment, for example, with the enzyme dispase (see U.S. Pat. No. 4,304,866). The cultured sheets can then be attached to petrolatum impregnated gauze or other non adherent backing, transported in culture medium to the operating room, and applied to the patient.
Large burn surface areas can be covered with autograft materials prepared by these methods, but the autografts require time to culture. While the cells for autografting are being cultured, it is possible to maintain the wound with allograft material which is effective as a temporary wound dressing. Allograft material also promotes healing of chronic skin ulcers and split-thickness graft donor sites. Cultured autograft material prepared by the method of Green, et al. are now available commercially from Biosurface Technology, Inc., of Cambridge, Mass. Allograft material is available for experimentation and clinical testing.
A severe, very practical limitation on the use of cultured epithelial grafts is their inherent limited shelf-life. The viability and colony-forming efficiency of the sheets falls rapidly after they are removed from the substrate on which they grow. This restricts the time and distance the sheets may be shipped from production facility to the operating room. The cell sheets are extraordinarily fragile. They are normally able to maintain their ability to resume growth and to form colonies when applied to wounds only for about six to eight hours or less after dispase release. These temporal limitations preclude maintenance of a large inventory. Development of a cryopreservation method for extending the storage interval of the cultured sheets would permit maintenance of large inventories for shipment throughout the world.
The art is replete with descriptions of various tissue preservation methods including cryopreservation, use of special cell media, and certain packaging techniques. Cryopreservation allows for long-term storage by freezing the material in the presence of a cryoprotective agent. This agent displaces the aqueous material in and around the cells and thereby prevents ice crystals from forming. Numerous disclosed protocols vary the nature or amount of cryoprotective agent, and/or the time, course, or the temperature of the freezing process in an attempt to retain cell viability after a freeze/thaw cycle. See, for example, U.S. Pat. No. 4,559,298, U.S. Pat. No. 4,688,387.
Storing tissue by means of cryopreservation is a complicated and expensive process, capable of yielding highly variable results. However, no other approach has been shown to extend storage viability of animal tissue beyond very short periods, i.e., eight hours. See, for example, Pittelkow et al., 86 J. Invest. Dermatol. 4: 410-17, 413-14 (1986). For years skin banks have used frozen, human skin on burns as temporary allograft coverage. However, this frozen skin is not truly viable. Although banked skin is metabolically active, it is uncertain whether the epidermal cells can reproduce themselves. Heimbach, D., et al., "Artificial Dermis for Major Burns: a Multi-Center Randomized Clinical Trial", Ann. Surg. 313-320 (September 1988).
Large scale production of cultured epithelial autografts, as described by Green et al, promotes permanent coverage of large surface area wounds with the patients own skin. Although large amounts of cultured epithelium can be produced for patients, the limited shelf-life of the epithelium is a major concern. To this end, Cancedda and DeLuca have developed a protocol whereby confluent sheets of cultured keratinocytes are frozen in culture medium containing 10% glycerol (see EP 0 296 475). However, experience with this procedure indicates that cell recovery is variable and generally very low. In addition, the narrow time ranges of incubation makes this method impractical in large scale production. Moreover, grafts frozen with any protocol and then thawed after storage in liquid nitrogen often crack. Although wound coverage may be possible, the actual condition of the confluent sheets at the cellular level remains in question.
Early work using high molecular weight cryoprotectants showed that polyvinylpyrrolidone (PVP) or dextrans (MW 30-100Kd) alone prevents destruction of erythrocytes during cryopreservation Pegg, D. E., "Banking of Cells, Tissues and Organs at Low Temperatures", in Current Trends in Cryobiology (A. Smith, ed. 1970). Dextran and hydroxyethyl starch combined with glycerol will partly maintain the motility of frozen and thawed ram spermatazoa Schmehl et al., "The Effects of Nonpenetrating Cryoprotectants Added to Test-Yolk-Glycerol Extender on the Post-Thaw Motility of Ram Spermatazoa" 23 Cryobiol. 6:512-17 (1986).
Using trypan blue dye exclusion as a measure of viability, hydroxyethyl starch (HES) alone has been demonstrated to improve the survival of cryopreserved cells of hematopoietic origin, but only if the cryoprotectant was added and later removed in a slow, time-consuming manner (Conscience and Fischer, "An Improved Preservation Technique for Cells of Hemapoetic Origin" 22 Cryobiol. 5:495-98 (1985). This parameter of viability provides information only about short-term membrane stability and offers no data about the proliferative potential or long-term survival of the tissue. These authors concluded that HES did not offer any advantage for cryopreservation of cells of epithelial origin. In another study, HES was successfully used in the cryopreservation of cells of hematopoietic origin as determined by cell proliferation assays, a parameter that is more reflective of long-term viability (Wang, et al, Cryobiol. 24:229-237 (1987).
Studies using high molecular weight, non-penetrating (glass-forming) agents as cryoprotectants tend to focus on cells in suspension such as red blood cells and lymphocytes as described above. However, the need to maintain the integrity of a cohesive sheet of cells during cryopreservation places severe limitations on the recovery of viable cells (i.e., those with the capability of tissue regeneration).
It is an object of this invention to provide a cryopreservation methodology, capable of preserving a living, cultured sheet of epithelial cells, where the method maintains the structural integrity of the sheet, and preserves the mitotic competence of cells in the sheet, to permit formation of an epithelial tissue useful in wound healing.